Y-shape branched hydrophilic polymer derivatives, their preparation methods, conjugates of the derivatives and drug molecules, and pharmaceutical compositions comprising the conjugates

ABSTRACT

The present invention relates to Y-shape branched PEG derivatives of formulae (I) to (IV). The present invention also relates to conjugates of these Y-shape derivatives and drug molecules, pharmaceutical compositions comprising those conjugates.

FIELDS OF THE INVENTION

The present invention relates to Y-shape branched hydrophilic polymerderivatives, their preparation methods, and conjugates of thederivatives and drug molecules, especially proteins and polypeptides.This invention also relates to pharmaceutical compositions comprisingthe conjugates.

BACKGROUND OF THE INVENTION

Natural and recombinant proteins and polypeptides have been used asmedicines. The products after purification and separation can be used totreat specific diseases by parenteral routes. When administeredparenterally, however, proteins may have immunogenicity, or berelatively insoluble in water, or have short pharmacological half-lives.How to raise and keep a highly effective serum concentration in vivo isof significant importance.

In addition to proteins, clinically there is necessity to modify otherconstituents of natural medicine such as flavonoids, terpenoids,anthraquinones, steroids and alkaloids to prolong their physiologicalhalf-lives, enhance their stability and the possibility to reach thetarget site, raise their solubility in water, change administrationroutes and improve bioavailability.

Recently PEG has been widely used to conjugate proteins, peptides orother therapeutic agents, in order to prolong their physiologicalhalf-lives and lower their immunogenicity and toxicity. Clinically, PEGand its derivatives have been widely used as carriers in the manufactureof pharmaceutical preparations of commercial drugs. The methods forconjugating PEG to drug molecules has made much progress in the last 10years and had been applied to many officially approved drugs. Forexample, PEG-intron®, a conjugate of PEG to α-interferon, exhibitslonger circulation half-life and better therapeutic effect. Theconjugate of PEG to paclitaxel reduces the toxicity and increases thebioactivity. The metabolism of PEG is well known, and PEG is accepted asa safe drug modifier.

One process called PEGylation is often applied when conjugating PEG todrugs. Namely, one or two of the terminal groups of the PEG areactivated to form a proper functional group, which is reactive to atleast one functional group of the drugs, and can form a stable bond withit.

Many PEG derivatives have been reported. Linear PEG propionic acid,butanoic acid and their NHS esters have been reported in U.S. Pat. No.5,672,662. Recently a U-shape branched PEG is reported in U.S. Pat. No.564,357. In these PEG derivatives, two linear PEGs link to one moleculeor structure through two identical functional groups, such as two aminogroups or two carboxyl groups. In one example of the patent, thebranched PEG is derived from linear PEG and lysine, which is a kind ofamino acid having two amino groups.

SUMMARY OF THE INVENTION

The present invention provides a new Y-shape branched hydrophilicpolymer derivative, which is represented by formula I:

wherein

-   P_(a) and P_(b) are hydrophilic polymers, which are the same or    different;-   j is an integer from 1 to 12;-   R_(i) is selected from the group consisting of H, a C₁₋₁₂    substituted or unsubstituted alkyl, a substituted aryl, an aralkyl,    and a heteroalkyl;-   X₁ and X₂ independently are linking groups, wherein X₁ is (CH₂)_(n),    and X₂ is selected from the group consisting of (CH₂)_(n),    (CH₂)_(n)OCO, (CH₂)_(n)NHCO and (CH₂)_(n)CO, wherein n is an integer    of from 1-10; and-   F is a functional group selected from the group consisting of a    hydroxyl group, a carboxyl group, an ester group, carboxylic acid    chloride, hydrazide, maleimide and pyridine disulfide, being capable    of reacting with an amino group, a hydroxyl group or a thiol group    of a therapeutic agent or a substrate to form a covalent linkage.

According to another aspect of the invention, there is provided aY-shaped branched poly(ethylene glycol) derivatives represented byformula II:

wherein

-   P_(a) and P_(b) are polyethylene glycols, which are the same or    different;-   n and j are independently an integer from 1 to 12;-   R_(i) is selected from the group consisting of H, a C₁₋₁₂    substituted or unsubstituted alkyl, a substituted aryl, an aralkyl,    and a heteroalkyl; and-   F is a functional group selected from the group consisting of a    hydroxyl group, a carboxyl group, an ester group, carboxylic acid    chloride, hydrazide, maleimide and pyridine disulfide, being capable    of reacting with an amino group, a hydroxyl group or a thiol group    of a therapeutic agent or a substrate to form a covalent linkage.

According to still another aspect of the invention, there is provided aY-shaped branched poly(ethylene glycol) derivatives represented byformula III:

wherein

-   P_(a) and P_(b) are polyethylene glycols, which are the same or    different;-   n, m and j are independently an integer from 1 to 12;-   R_(i) is selected from the group consisting of H, a C₁₋₁₂    substituted or unsubstituted alkyl, a substituted aryl, an aralkyl,    and a heteroalkyl; and-   F is a functional group selected from the group consisting of a    hydroxyl group, a carboxyl group, an ester group, carboxylic acid    chloride, hydrazide, maleimide and pyridine disulfide, being capable    of reacting with an amino group, a hydroxyl group or a thiol group    of a therapeutic agent or a substrate to form a covalent linkage.

According to still another aspect of the invention, there is provided aY-shaped branched poly(ethylene glycol) derivatives represented byformula IV:

wherein

-   P_(a) and P_(b) are polyethylene glycols, which are the same or    different;-   n and j are independently an integer from 1 to 12;-   R_(i) is selected from the group consisting of H, a C₁₋₁₂    substituted or unsubstituted alkyl, a substituted aryl, an aralkyl,    and a heteroalkyl; and-   F is a functional group selected from the group consisting of a    hydroxyl group, a carboxyl group, an ester group, carboxylic acid    chloride, hydrazide, maleimide and pyridine disulfide, being capable    of reacting with an amino group, a hydroxyl group or a thiol group    of a therapeutic agent or a substrate to form a covalent linkage.

According to still another aspect of the invention, there is provided amethod to prepare the PEG derivative of formula II, comprising:

at 0° C. initiating the polymerization of ethylene oxide withN,N-di-2-hydroxylethyl-2-benzyloxyethyl amine in the presence of acatalyst;

alkylating terminal hydroxyl groups;

removing benzyl groups by catalytic hydrogenation; and

derivatizing the new hydroxyl group to incorporate the terminal group F.

According to still another aspect of the invention, there is provided amethod to prepare the PEG derivatives of formulae (II) and (III),comprising:

reacting one methoxyl polyethylene glycol mesylate with an amino acidunder basic conditions,

reacting the product obtained above with another methoxyl polyethyleneglycol derivative, and further derivatizing to incorporate a terminalgroup F.

According to still another aspect of the invention, there is providedconjugates of the above polymer derivatives and drug molecules throughthe functional group F.

According to still another aspect of the invention, there is providedpharmaceutical compositions comprising the above conjugates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of Y-shape branched polyethylene glycolderivatives (1).

FIG. 2 shows the synthesis of Y-shape branched polyethylene glycolderivatives (2) and (7).

FIG. 3 shows the synthesis of Y-shape branched polyethylene glycolderivatives (5).

FIG. 4 shows the synthesis of Y-shape branched polyethylene glycolderivatives (6).

FIG. 5 shows the synthesis of conjugates of Y-shape branchedpolyethylene glycol derivatives (1) and drugs (through ester bonds).

FIG. 6 shows the synthesis of conjugates of Y-shape branchedpolyethylene glycol derivatives and drugs (through other bonds).

FIG. 7 shows the synthesis of conjugates of Y-shape branchedpolyethylene glycol derivatives and proteins.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the hydrophilic polymer is, for example, apolyethylene glycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine or a copolymer thereof, especially preferred are apolyethylene glycol and copolymers thereof.

In the PEG derivatives of formulae (II) to (IV) of the presentinvention, Pa and Pb may be the same or different, and can be the PEGrepresented by the following formula (V):

wherein:

-   R is H, a C₁₋₁₂ allyl, cycloalkyl or aralkyl; and-   n is an integer, representing the degree of polymerization,    preferably making the molecular weight of PEG is 300 to 60000.

In formula (V), R is preferably H, methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclohexyl or benzyl.

The Y-shaped branched hydrophilic polymer derivatives of the presentinvention are preferably prepared by attaching two linear PEG chains toan amino group of a small molecule.

PEG is used here as an example to illustrate the preparation of Y-shapedbranched hydrophilic polymer derivatives of the present invention.

The general structure of PEG is as the formula below:

wherein:R is H, a C₁₋₁₂ alkyl, a cycloalkyl base or an aralkyl, andn is an integer, representing the degree of the polymerization.

As a lower alkyl, R can be any lower alkyl group having 1-6 carbonatoms, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, n-pentyl, or n-hexyl. As a cycloalkyl, R is preferably acycloalkyl containing 3-7 carbon atoms, for example, cyclopropyl,cyclobutyl, and cyclohexyl. Among those, cyclohexyl is more preferable.The typical compound is methoxy-polyethylene glycol (mPEG). Otheranalogs and derivatives of polyethylene glycol, such as polypropyleneglycols, polyvinyl alcohols, and polyacrylmorpholines and the like, canalso be used in the present invention.

In respect of PEGs, they are usually measured by molecular weight. It ispreferred that the molecular weight of PEG which forms the conjugatesfalls in the range from 300 to 60000 Daltons, which means n is about 6to 1300. It is more preferred that n is 28, 112 and 450, respectivelycorresponding to molecular weight of 1325, 5000, and 20000. Because ofthe potential non-homogeneity of the starting

PEGs which are usually defined by their, molecular weights rather thanthe self-repeating unit n, PEGs are normally characterized with a weightaverage molecular weight, rather than their self-repeating unitsrepresented by n. The starting PEG compounds with different molecularweights are readily synthesized using methods known in the art or theyare commercially available.

The Y-shape PEG derivatives of the present invention can be synthesizedand prepared by general methods in this field. The different compoundsclaimed in the invention are synthesized and prepared by known methods,which can be found in the technological literatures and patents in thisfield.

For Pa and Pb being mPEGs, X₁ and X₂ being a simple branched alkyl, andF being a hydroxyl group, the possible formula is below:

This compound can be prepared by using a standard polymerizationinitiator, to promote the polymerization of ethylene oxide or ethyleneglycol. A standard method of preparation is shown below:

For P_(a) and P_(b) being methoxy polyethylene glycols (mPEGs), X₁ andX₂ being different, the possible formula is below:

This compound can be obtained from stepwise reactions of a PEG with acompound containing an amino group. The selected compound containing anamino group can be an amino acid, an amino ketone or another moleculehaving an amino group. The standard preparation route is illustratedbelow. However, there are other standard methods useful for preparingthis derivative that are known in this field.

When the hydrophilic polymer derivatives of present invention are used,the F terminal group plays a key role. Derivatives with differentterminal groups have different uses. The introduction of thesefunctional groups determines the applicable fields and structures ofthese derivatives. In respect of the desired use, the following methodscan be used to modify the terminal functional group:

1. Amination

Because of greater reactivity of amino groups over hydroxyl groups, theaminated PEG derivatives are important in reacting with a moleculehaving a carboxylic acid group to yield a conjugate.

2. Carboxylation

Carboxylation helps to improve PEG's reactivity and makes it capable ofconjugating to molecules having amino or hydroxyl groups.

If an amino acid is used as a starting material, the terminal group ofthe resulting Y-shaped PEG will have a carboxylic group. Especially, ifmultiple carboxylic acid containing amino acids or polymers are used,the terminal groups will have several carboxylic acid groups. This kindof structure is useful to increase the load of small natural drugmolecule and achieve a slow-release effect by stepwise degradation.

3. Other Modification Methods

Other modification, for example, by acid chloride, hydrazine, maleimide,pyridine disulfide and the like can be appropriately adopted as well toobtain corresponding derivatives. Other preparation methods in thisfield will be apparent to those skilled in this are.

Many components of natural drugs have active functional groups such asamino, carboxyl and hydroxyl groups, which bind with monosaccharides,polysaccharides, nucleosides, polynucleosides, phosphoryl and the likein vivo, to form active pharmacological structures.

Similarly, the PEG derivatives with the modified terminal functionalgroup can conjugate to the drug molecules in the same way to take theplace of a bio-organic molecule and overcome the shortcomings of shortphysiological half-lives and low therapeutic effect. The following modelis a general ester synthesis reaction.

An ester group can be eliminated by biodegradation in vivo to releasethe active ingredient. An amide group is relatively stable in vivo.

The Y-shape hydrophilic polymer derivatives of the present invention canform conjugates with drug molecules through proper functional groups.These functional groups can link the free amino, hydroxyl or thiol groupof proteins, polypeptides and other natural drugs with the polymerderivatives. For proteins and peptides with high molecular weight, onemolecule can link with one or more PEG derivatives to improve thephysiological effects of the drug molecules in vivo. For the activecomponents of a natural drug with low molecular weight, one PEGderivative can be linked to one or more drug molecules through properfunctional groups to ensure a proper drug concentration and the functionof sustained release.

The applications described above offer some possible reference modelsfor medical application of the PEG derivatives. The choice of properderivatives for real applications can be confirmed by using animalpharmacology, toxicology, clinical study and other such approaches.

Preferably, the drug molecules included in the conjugates of the presentinvention are the active ingredients separated from nature plants, suchas paclitaxel, camptothecin, cinobufagin, clycyrrhetinic acid andscopoletin. Preferably, the drugs are the ingredients of naturalmedicines used in the treatment of tumors, such as paclitaxel,camptothecin, and derivatives thereof. Preferred drugs also includeinterferons, such as α- and β-interferon.

The conjugates of the present invention can be administered in the formof pure compounds or suitable pharmaceutical compositions, via anyacceptable routes or being included in a reagent for similar use. Thus,the conjugates can be administered via oral, nasal, parenteral, topical,transdermal, rectal or injection routes in the form of solid, semisolid,lyophilized powder or liquid, for example, tablets, suppositories,pills, soft and hard gelatin capsules, powder, solution, suspention andaerosols. Preferably the unit dosage form is suitable for aprecise-dosage and easy administration. The composition includesconventional pharmaceutical carriers or excipients and the conjugate(s)of the present invention as the active ingredient(s). Furthermore, italso can include other agents, carriers and excipients.

Generally speaking, depending on the method of administration, thepharmaceutically acceptable compositions will include about 1-99 wt. %of the conjugate of the present invention, and 99-1 wt. % of suitablepharmaceutical excipient. Preferably they include 5-75 wt. % of theconjugate and the rest is any suitable pharmaceutical excipient.

The preferable way of administration is injection with a general dailydosage scheme, which can be adjusted based on the severity of thedisease to be treated. The conjugates of the present invention or theirpharmaceutically acceptable salts may be formulated in the dosage forinjection by, for example, dissolving 0.5-50% of the active componentsin a liquid pharmaceutical carrier, such as water, saline, aqueousglucose, glycerol, ethanol and the like to form a solution ofsuspension.

The compositions which can be administered as liquid such as solutionsand suspensions can be prepared by dissolving and dispersing theconjugate of the present invention (about 0.5-20%) and optionally a thepharmaceutical excipient into a carrier. Example of carriers includeswater, saline, aqueous glucose, glycerol, ethanol and the like.

If needed, the pharmaceutical composition of the present invention canfurther include an adjuvant in a small amount, such as wetting agent,emulsifier, pH buffer, antioxidant and the like. For example, citricacid, sorbitan monolaurate, triethanolamine oleate, butylatedhydroxytoluene and the like can be added.

The practical preparation methods of such dosage forms are known orobvious to the skilled in the art. For example, see Ramington'sPharmaceutical Sciences, 18^(th) edition, (Mack Publishing Company,Easton, Pa., 1990). In any case, according to the techniques of thepresent invention, the composition applied will include an effectiveamount of the conjugate of the present invention for the treatment ofcorresponding disease.

EXAMPLES

The polymer derivatives and the conjugates of the present invention andtheir preparation methods will be further described by the followingexamples. These examples do not intend to limit the scope of theinvention by any means. The scope of the present invention can bedetermined by the claims.

Example 1 Synthesis of Y-Shape Branched Polyethylene Glycol Derivatives(1)

The synthesis is shown in FIG. 1. At 0° C., 10 ml of dry ethylene oxidewas added to a heavy-duty flask, which included 200 mg ofN,N-di-2-hydroxylethyl-2-benzyloxyethyl amine and 50 mg of dry NaH. Thereaction mixture was stirred with the temperature being slowlyincreased. After 28 hours, the viscous liquid was quenched with waterand the polymer was extracted with dichloromethane. The organic phasewas dried with anhydrous sodium sulfate, and the solvent was removedunder vacuum. Yield: 8.2 g (81%), Mp: 56-58° C.

5 g of (PEG)₂-N—CH₂CH₂O-Bz (molecular weight is 10000, obtained fromabove step) was dissolved in 50 ml toluene. 0.1 g of sodium hydride and0.5 g of benzene sulfonic acid methyl ester were added to the solution.The reaction mixture was heated at 80° C. for 24 hours. Then thesolution was quenched with 2 ml of isopropyl alcohol. The solvent wasremoved under vacuum and the residue was added to 200 ml of isopropylalcohol. The precipitate was collected by filtration and dried invacuum. Yield: 4.6 g (92%), Mp: 57-59° C.

3 g of (MeO-PEG)₂-N—CH₂CH₂O-Bz (molecular weight is 10000, obtained fromabove step) was dissolved in 30 ml of anhydrous 1,4-dioxane. Then, 0.1 gof Pd/C was added to the solution mixture as catalyst and H₂ gas (40psi) was introduced into the reactor. The solution was vigorouslystirred at room temperature overnight. The catalyst was removed byfiltration and was washed with fresh dichloromethane. The solvent wasremoved by rotary evaporation and the residue was added into ethylether. The precipitate was collected by filtration and dried in vacuum.Yield: 2.4 g (80%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s, 6H), 2.63(t, 6H).

Example 2 Synthesis of Y-Shape Branched Polyethylene Glycol SuccinimidylCarbonate (2)

The synthesis is shown in FIG. 2. 1 g of Y-shape branched PEG derivativeof Mw 10000 ((MeO-PEG)₂-N—CH₂CH₂OH, from example 1) and 0.1 g ofdi-succinimidyl carbonate were dissolved in 20 ml of acetonitrile. 0.1ml of pyridine was added to the solution. The reaction mixture wasstirred under the protection of nitrogen overnight. The solvent wasremoved by rotary evaporation and the residue was dried under vacuum.The solid residue was added to 10 ml of dry dichloromethane. Theundissolved solid was filtered. The organic phase was washed with sodiumacetate buffer (0.1M, pH 5.5), dried with anhydrous sodium sulfate,concentrated by rotary evaporation, and precipitated in ethyl ether. Theproduct was dried in vacuum. Yield: 0.9 g (90%). NMR (DMSO): 3.5 (br m,H in PEG), 3.24 (s, 6H), 4.45 (t, 2H), 2.82 (s, 4H)

Example 3 Synthesis of mPEG-Glycine (3)

5 g mPEG of molecular weight 5000 was dissolved in 50 ml toluene,azeotropically distilled for 2 hours under the protection of nitrogen,with 10 ml solution being distilled off, and then cooled to roomtemperature. 3 ml of dry dichloromethane and 0.08 ml dry triethylaminewere added to the reaction. The mixture was cooled in an ice-water bathand 0.12 ml of dry methanesulfonyl chloride was added dropwise. Themixture was stirred at room temperature under the protection of nitrogenovernight. The reaction was quenched by adding 2 ml of absolute ethanol.Part of the solvent was removed by rotary evaporation, the precipitatewas collected by filtration, and then 150 ml ethyl ether was added. Theprecipitate was collected by filtration and dried in vacuum. Yield: 4.8g (96%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s, 3H), 4.32 (t, 2H).

2 g of glycine hydrochloride was dissolved in 20 ml of deionized water.1 g of NaOH was added to the glycine solution to adjust the pH to 10.5.Then 2 g of mPEG mesylate ester of molecular weight 5000 (obtained fromabove step) was added to the solution. The solution was incubated at 37°C. for 72 hours, and then neutralized by hydrochloride solution to pHabout 7. The polymer was extracted with dichloromethane. The organicphase was dried with anhydrous sodium sulfate and the solvent wasremoved under vacuum. Yield: 1.7 g (85%), Mp: 55-57° C. NMR (DMSO): 3.5(br m, H in PEG), 3.24 (s, 31-1), 2.95 (t, 2H), 3.11 (s, 2H).

Example 4 Synthesis of mPEG-Alanine (4)

5 g mPEG of molecular weight 5000 was dissolved in 50 ml of toluene,azeotropically distilled for 2 hours under the protection of nitrogenwith 10 ml solvent being distilled off, and then cooled to roomtemperature. 3 ml of dry dichloromethane and 0.08 ml of drytriethylamine were added to the reaction. The mixture was cooled in anice-water bath and 0.12 ml dry methanesulfonyl chloride was addeddropwise. The mixture was stirred at room temperature under theprotection of nitrogen overnight. The reaction was quenched by adding 2ml of absolute ethanol. The solvent was removed by rotary evaporation,the precipitate was collected by filtration, and then 150 ml ethyl etherwas added. The precipitate was collected by filtration and dried invacuum. Yield: 4.5 g (90%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s,3H), 4.32 (t, 2H).

2 g of alanine hydrochloride was dissolved in 20 ml of deionized water.1 g of NaOH was added to the alanine solution to adjust the pH to 10.5.Then 2 g of mPEG mesylate of molecular weight 5000 was added to thesolution. The solution was incubated at 37° C. for 72 hours, and thenneutralized with hydrochloride solution to pH about 7. The polymer wasextracted with dichloromethane. The organic phase was dried withanhydrous sodium sulfate, and the solvent was removed under vacuum.Yield: 1.9 g (94%), Mp: 55-57° C. NMR (DMSO): 3.5 (br m, H in PEG), 3.24(s, 3H), 2.94 (m, 1H), 1.24 (d, 3H).

Example 5 Synthesis of Y-Shape Branched PEG Derivatives (5) Reactive toan Amino Group

The synthesis is shown in FIG. 3. 1 g of mPEG-glycine (3) ormPEG-alanine (4) of molecular weight 5000 (from example 3 or 4) wasdissolved in 20 ml of dichloromethane. 1 g of mPEG carboxyethyl NHSester (mPEG-O—CH₂—CO—NHS, molecular weight 5000) and 0.1 ml oftriethylamine were added to the solution. The solution was stirredovernight. The solvent was removed under vacuum and the residue wasadded to ethyl ether. The precipitate was collected by filtration anddried under vacuum. The product (V-shape branched PEG acid) was furtherpurified by ion exchange chromatography. Yield: 0.98 g (50%).

0.5 g of Y-shape branched mPEG acid was dissolved in 10 mldichloromethane. 7 mg of N-hydroxylsuccinimide (NHS) and 13 mg ofdicyclohexylcarbodiimide (DCC) was added to the solution. The solutionwas stirred at room temperature for 6 hours. The solvent was removedunder vacuum. The residue was added to 20 ml of isopropyl alcohol (EPA).The product was collected by filtration and dried under vacuum. Yield:0.48 g (96%), NMR (DMSO): 3.5 (br m, H in PEG), 3.24, (s, 6H), 2.81 (s,4H), 4.15 (s, 2H), 4.07 (t, 2H), 4.48 (t, 2H).

Example 6 Synthesis of Y-Shape Branched PEG Derivatives (6) ReactiveToward Amine Group

The synthesis is shown in FIG. 4. 1 g of mPEG-glycine (3) ormPEG-alanine (4) of molecular weight 5000 (from example 3 or 4) wasdissolved in 20 ml of dichloromethane. 1 g of mPEG NHS carbonate(mPEGO-CO—NHS) of Mw 5000 Dalton and 0.1 ml of triethylamine were addedto the solution. The solution was stirred overnight. The solvent wasremoved under vacuum and the residue was added to ethyl ether. Theprecipitate was collected by filtration and dried under vacuum. Theproduct (Y-shape branched PEG acid) was further purified by ion exchangechromatography. Yield 0.98 g (50%)

0.5 g of Y-shape branched mPEG acid was dissolved in 10 mldichloromethane. 7 mg of N-hydroxylsuccinimide (NHS) and 13 mg ofdicyclohexylcarbodiimide were added to the solution. The solution wasstirred at room temperature for 6 hours. The solvent was removed undervacuum. The residue was added to 20 ml of isopropyl alcohol. Theprecipitate was collected by filtration and dried under vacuum. Yield0.48 g (96%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s, 6H), 2.81 (s,4H), 4.15 (s, 2H), 4.07 (t, 2H).

Example 7 Synthesis of Y-Shape Branched PEG Derivatives (7) ReactiveToward Thiol Group

The synthesis is shown in FIG. 2. 1 g of Y-shape branched PEG((MeO-PEG)₂-NCH₂CH₂OH) of molecular weight 10000 (obtained in Example 2)was dissolved in toluene, azeotropically distilled for 2 hours underprotection of nitrogen, and then cooled to room temperature. 3 ml of drydichloromethane and 0.08 ml dry triethylamine were added to thesolution. The mixture was cooled in an ice-water bath and drymethanesulfonyl chloride was added dropwise. The mixture was stirred atroom temperature under dry nitrogen overnight. The reaction was quenchedby adding 3 ml of absolute ethanol. The solvent was removed by rotaryevaporation, the precipitate was removed by filtration, and then 150 mlof ethyl ether was added. The precipitate was collected by filtrationand dried in vacuum. Yield: 0.8 g (80%)

1 g of Y-shape branched PEG mesylate ((MeO-PEG)₂-N—CH₂CH₂OMs) ofmolecular weight 10000 was dissolved in 30 ml of aqueous ammoniasolution with 5% ammonium chloride. The solution was stirred over 72hours at room temperature. The solution was extracted withdichloromethane three times. The combined organic phase was dried withanhydrous sodium sulfate. The solvent was removed under vacuum. Theresidue was added to 50 ml isopropyl alcohol. The precipitate wascollected and dried under vacuum. Yield: 0.7 g (70%)

0.5 g of Y-shape branched PEG amine((MeO-PEG)₂-N—CH₂CH₂NH₂) wasdissolved in acetonitrile. 20 mg of NHS-3-maleimidopropionate was addedto the solution. The solution was stirred overnight at room temperature.The solvent was removed under vacuum. The residue was added to 30 mlisopropyl alcohol. The precipitated was collected and dried undervacuum. Yield: 0.42 g (84%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s,6H), 3.05 (t, 2H), 2.56 (t, 2H), 6.71 (s, 2H in maleimide).

Example 8

Conjugate of Y-shape Branched PEG-NHS Derivatives with α-Interferon (8)

The synthesis is shown in FIG. 7. 75 mg Y-shape branched polyethyleneglycol succinimidyl ester (from example 2, 5 or 6) was dissolved in 5 mlof buffered α-interferon solution with interferon concentration 5 mg/ml(pH 7.4). In the reaction solution PEG and α-interferon were at ratio of3:1. The solution was gently shaken for 1 hour at 4° C. and then 5 hoursat room temperature. The solution was diluted to a final interferonconcentration of 0.5 mg/ml and purified by HPLC with gel column. Themono-substituted Y-shape branched PEG conjugate of α-interferon wascollected. SDS-PAGE showed the product contained no free α-interferon.

SDS-PAGE Analysis: Reaction mixture and the purified PEG-IFN wassubjected to sodium dodecyl(lauryl) sulfate/polyacrylamide (8-16%) gelelectrophoresis and stained for protein using Coomassie blue dye. PEGmoieties in the PEG2-IFN conjugates were specifically stained usingTitrisol iodine solution (EM Science, Gibbstown, N.J.). The SDS-PAGE gelwas rinsed with distilled water and placed in 5% barium chloridesolution. After 10 min, the above gel was washed with distilled waterand placed in 0.1 N Titrisol iodine solution for another 10 min.Titrisol was washed off with distilled water. The PEG stained (orangebrown bands) SDS-PAGE gel containing Y-PEG-IFN samples was stored indistilled water in a heat-sealed Kapak/Scotchpak bag.

Example 9 Conjugate of Y-shape branched PEG-NHS derivatives withβ-interferon

Y-shape branched polyethylene glycol succinimidyl ester (Example 5 or 6)was dissolved in 5 ml of buffered β-interferon solution with interferonconcentration of 1 mg/ml (pH 7.4). In the reaction solution, PEG andβ-interferon were at ratio of 3:1. The solution was gently shaken for 7hours. The solution was purified by HPLC with gel column. Themono-substituted Y-shape branched PEG conjugate of β-interferon wascollected. SDS-PAGE and CE showed the product contains no freeβ-interferon.

Example 10

Conjugate of Y-Shape Branched PEG Derivatives with Paclitaxel (10)

The synthesis is shown in FIG. 5. 1 g of Y-shape branched PEG carboxylicacid (from Example 5 or 6) was dissolved in 10 ml dichloromethane. 90 mgof paclitaxel, 8 mg of dimethylamino pyridine and 25 mg ofdicyclohexylcarbodiimide were added to the solution. The solution wasstirred at room temperature for 6 hours. The solvent was removed undervacuum. The residue was added to 20 ml of isopropyl alcohol. Theprecipitate was collected by filtration, washed with ether and driedunder vacuum. Yield: 0.8 g (80%), Mp: 55-57° C.

Example 11 Conjugate of Y-Shape Branched PEG Derivatives withCamptothecin (11)

The synthesis is shown in FIG. 6. 1 g of Y-shape branched PEG carboxylicacid (Example 5 or 6) was dissolved in 10 ml dichloromethane. 120 mg ofglycine-camptothecin, 50 mg of dimethylamino pyridine and 95 mg ofdicyclohexylcarbodiimide were added to the solution. The solution wasstirred at room temperature for 6 hours. The solvent was removed undervacuum. The residue was dissolved in 20 ml of 1,4-dioxane. Theprecipitate was removed by filtration. The solution was concentrated,and the residue was added to 20 ml of ethyl ether. The precipitate wascollected by filtration, washed with ethyl ether and dried under vacuum.Yield: 0.8 g (80%), Mp: 56-58° C.

Example 12 Conjugate of Y-Shape Branched PEG Derivatives withCinobufagin (12)

The synthesis is shown in FIG. 5. 1 g of Y-shape branched PEG carboxylicacid (Example 5 or 6) was dissolved in 10 ml dichloromethane. 60 mg ofCinobufagin, 12 mg 1-Hydroxybenzotriazole, 16 mg of dimethylaminopyridine and 40 mg of dicyclohexyl-carbodiimide were added to thesolution. The solution was stirred at room temperature for 6 hours. Thesolvent was removed under vacuum. The residue was added to 20 ml ofisopropyl alcohol. The precipitate was collected by filtration, washedwith ether and dried under vacuum. Yield: 0.75 g (75%), Mp: 57-59° C.

Example 13

Conjugate of Y-Shape Branched PEG Derivatives with Scopoletin (13)

The synthesis is shown in FIG. 5. 1 g of Y-shape branched PEG carboxylicacid (Example 5 or 6) was dissolved in 20 ml of dichloromethane. 30 mgof Cinobufagin, 20 mg of 1-Hydroxybenzotriazole, 20 mg of dimethylaminopyridine and 38 mg of dicyclohexyl-carbodiimide were added to thesolution. The solution was stirred at room temperature for 12 hoursunder the protection of nitrogen. The solvent was concentrated undervacuum. The residue was added to 20 ml of 1,4-dioxane. The precipitatewas collected by filtration, washed with ether and dried by air exhaust.The solvent was removed under vacuum. The remaining residue was added to100 ml of isopropyl alcohol. The precipitate was collected byfiltration, washed with ether and dried by air exhaust. The precipitateswere combined and dried under vacuum. Yield: 0.92 g (92%), Mp: 56-58° C.

Example 14 Conjugate of Y-Shape Branched PEG Derivatives withClycyrrhetinic Acid (14)

The synthesis is shown in FIG. 6. 1 g of Y-shape branched PEG carboxylicacid (Example 5 or 6) was dissolved in 10 ml of dichloromethane. 0.2 mlof thionyl chloride was added to the solution. The solution was stirredfor 2 hours. The solvent and impurities having low boiling point wereremoved under vacuum. 10 ml of dichloromethane solution having 70 mgclycyrrhetinic acid was added, and dissolved by mixing. Then 60 mg4-dimethylamino pyridine was added. The reaction mixture was stirred for12 hours at room temperature under the protection of nitrogen gas. Thesolvent was concentrated under vacuum. The residue was added into 20 mlof isopropyl alcohol. The precipitate was collected by filtration,washed with ethyl ether, dried by air exhaust, and further dried undervacuum. Yield: 0.6 g (60%). M.p.: 58˜60° C.

Example 15

This example is to illustrate the preparation process of a typicalpharmaceutical composition administered parenterally. The compositioncomprises the conjugate of the present invention.

Component Conjugate prepared in Example 8 2 g 0.9% saline 100 ml

The conjugate prepared in Example 8 was dissolved in 0.9% saline toobtain 100 ml solution for intravenous injection, which was filteredthrough 0.2 μm membrane and packed aseptically. The powder for injectionwas obtained by freeze-drying.

1. (canceled)
 2. The hydrophilic polymer derivative of claim 4 whereinthe hydrophilic polymer is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, polyvinyl alcohol,polyacrylmorpholine and copolymers thereof.
 3. The hydrophilic polymerderivative of claim 2 wherein the hydrophilic polymer is polyethyleneglycol.
 4. A Y-shaped branched hydrophilic polymer derivativerepresented by formula II:

wherein P_(a) and P_(b) are hydrophilic polymers, which are the same ordifferent; n and j are independently an integer from 1 to 12; R_(i) isselected from the group consisting of H, a C₁₋₁₂ substituted orunsubstituted alkyl, a substituted aryl, an aralkyl, and a heteroalkyl;and F is a functional group capable of reacting with an amino group, ahydroxyl group or a thiol group of a therapeutic agent or a substrate toform a covalent linkage, selected from the group consisting of ahydroxyl group, a carboxyl group, an ester group, carboxylic acidchloride, hydrazide, maleimide and pyridine disulfide. 5-6. (canceled)7. The derivative of claim 4, wherein P_(a) and P_(b) are the same ordifferent polyethylene glycols (PEGs) of formula (V):

wherein R is H, a C₁₋₁₂ alkyl, a cycloalkyl or an aralkyl; and n is aninteger, representing the degree of polymerization.
 8. The derivative ofclaim 7, wherein R is selected from the group consisting of H, methyl,ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclohexyl and benzyl.
 9. Thederivative of claim 7, wherein the molecular weight of PEG is from about300 to
 60000. 10. A method to prepare the hydrophilic polymer derivativeof claim 4, comprising: initiating the polymerization of ethylene oxidewith N,N-di-2-hydroxylethyl-2-benzyloxyethyl amine in the presence of acatalyst; alkylating terminal hydroxyl groups; and removing benzylgroups by catalytic hydrogenation; and 11-12. (canceled)
 13. A conjugateformed by reacting the derivative of claim 4 with a drug moleculethrough the terminal group F.
 14. (canceled)
 15. The conjugate of claim13 wherein the drug is selected from the group consisting of aminoacids, proteins, enzymes, nucleosides, saccharides, organic acids,glycosides, flavonoids, anthraquinones, terpenoids, phenylpropanoidphenols, steroids, glycoside of the steroids and alkaloids of thesteroids.
 16. The conjugate of claim 13 wherein the drug is an activecomponent of a natural medicine.
 17. The conjugate of claim 16 whereinthe active component is cinobufagin, clycyrrhetinic acid or scopoletin.18. The conjugate of claim 13 wherein the drug is an anti-tumor agent.19. The conjugate of claim 18 wherein the anti-tumor agent is selectedfrom the group consisting of paclitaxel, camptothecin, interferon andderivatives thereof.
 20. The conjugate of claim 19 wherein theinterferon is α-, β- or γ-interferon.
 21. A pharmaceutical compositioncomprising the conjugate of claim 13 and optionally a pharmaceuticallyacceptable carrier and excipient.
 22. The method of claim 10, furthercomprising derivatizing to incorporate the terminal group F of claim 4.